Beta(β)-glucan is a complex carbohydrate, generally derived from several sources, including yeast, bacteria, fungi and cereal grains. Each type of β-glucan has a unique structure in which glucose is linked together in different ways, resulting in different physical and chemical properties. For example, β(1-3) glucan derived from bacterial and algae is linear, making it useful as a food thickener. The frequency of side chains, known as the degree of substitution or branching frequency, regulated secondary structure and solubility. Beta glucan derived from yeast is branched with β(1-3) and β(1-6) linkages, enhancing its ability to bind to and stimulate macrophages. β(1-3;1-6) glucan purified from baker's yeast (Saccharomyces cerevisiae) is a potent anti-infective beta-glucan immunomodulator.
The cell wall of S. cerevisiae is mainly composed of β-glucans, which are responsible for its shape and mechanical strength. While best known for its use as a food grade organism, yeast is also used as a source of zymosan, a crude insoluble extract used to stimulate a non-specific immune response. Yeast-derived beta (1,3;1,6) glucans stimulate the immune system, in part, by activating the innate anti-fungal immune mechanisms to fight a variety of targets. Baker's yeast β(1-3;1-6) glucan is a polysaccharide composed entirely of β(1-3)-linked sugar (glucose) molecules forming the polysaccharide backbone with periodic β(1-3) branches linked via β(1-6) linkages). It is more formally known as poly-(1-6)-β-D-glucopyranosyl-(1-3)-β-D-glucopyranose. Glucans are structurally and functionally different depending on the source and isolation methods.
Beta glucans possess a diverse range of activities. The ability of β-glucan to increase nonspecific immunity and resistance to infection is similar to that of endotoxin. Early studies on the effects of β(1,3) glucan on the immune system focused on mice. Subsequent studies demonstrated that β(1,3) glucan has strong immuno stimulating activity in a wide variety of other species, including earthworms, shrimp, fish, chicken, rats, rabbits, guinea pigs, sheep, pigs cattle and humans. Based on these studies, β(1,3) glucan represents a type of immunostimulant that is active across the evolutionary spectrum, likely representing an evolutionarily innate immune response directed against fungal pathogens. However, despite extensive investigation, no consensus has been achieved on the source, size, and form of β(1-3) glucan ideal for use as an immunostimulant.
The potential antitumor activity of β-glucans has been under investigation for about 30 years, as disclosed primarily in the Japanese pharmaceutical literature. Lentinan, for example, has been extensively investigated both in animal models at 1 mg/kg for 10 days and in clinical trials since the late 1970s for advanced or recurrent malignant lymphoma and colorectal, mammary, lung and gastric cancers. A recent review describes much of this work, which has focused on β-glucans isolated from mushrooms (Borchers, A T., et al., Mushrooms and Immunity, 221(4), 281 (1999)). This work indicates that the antitumor activity of polysaccharides isolated from mushrooms is largely mediated by T cells and macrophages, which are activated by β-glucan. Oral β-glucan isolated from crude yeast and cereal grain preparations has demonstrated antitumor activity as well. These studies used crude β(1,3) glucan preparations that are mixtures of β(1,3) glucan along with other polysaccharides such as β-glucans, mannans, chitin/chitosan, β(1,4) glucans, nucleic acids, proteins, and lipids. The β(1,3) glucan content of these preparations is typically less than 50% by weight. The effectiveness of various glucans differs in their ability to elicit various cellular responses, particularly cytokine expression and production, and in their activity against specific tumors. It has been proposed that the antitumor mechanism of action of β-glucans involves macrophage simulation and subsequent release of inflammatory mediators such as IL-1, TNF, and prostaglandin E2 (Sveinbjørnsson et al., Biochem. Biophys. Res. Commun. 223(3), 643 (1996)).
The immune system comprises two overall systems; the adaptive immune system and the innate immune system. β-glucans are considered to operate primarily through the relatively non-specific, innate immune system. The innate immune system includes complement proteins, macrophages, neutrophils, and natural killer (NK) cells, and serves as a rapid means of dealing with infection before the adaptive immune system can be brought to bear. Particulate β-glucan and high molecular weight soluble β-glucans such as lentinan and schizophyllan have been shown to be large enough to cross-link membrane CR3 of neutrophils and macrophages, triggering respiratory burst, degranulation, and cytokine release in the absence of target cells. (G. D. Ross, et al., Immunopharmacology 42, 61 (1999)). Neutral soluble β-glucan, on the other hand, does not simulate cytokine release, most likely because it is too small to cross-link membrane CR3.
The subtle changes associated with cancer development can lead to different expression of surface proteins, which can stimulate a weak response by the adaptive immune system. These changes in surface antigen expression also provide a target for treatment using selective monoclonal antibodies (mAbs) or antitumor vaccines. Monoclonal antibodies have been developed to target various proteins expressed in colon cancer, lymphoma, breast cancer, and acute leukemia, for example. The immune basis of the clinical tumor response to mAb includes direct cytotoxicity and induced immunity, in which antibody-dependent cell-mediated cytotoxicity and complement-mediated cytotoxicity are responsible for the direct killing of tumor cells. However, it has been noted that the increased complement activation mediated by natural or monoclonal antibodies often shows little effect on tumor growth due to the inherent resistance of tumors to complement-mediated cytotoxicity. This inherent resistance results in mAbs or vaccines to tumor antigens ineffective therapeutically. Monoclonal antibody (mAb) therapy is limited by effector mechanisms (e.g., antagonism of growth factor receptors, antibody-dependent cell-mediated cytotoxicity).
Tumor immunotherapy with humanized monoclonal antibodies (mAbs) such as Herceptin™ (trastuzumab) and Rituxan™ (rituximab) is now accepted clinical practice in patients with Her-2/neu+ metastatic mammary carcinoma and B cell lymphoma, respectively (Wang, S. C., et. al., Semin. Oncol., 28: 21-29, 2001; Leyland-Jones, B., Lancet Oncol., 3: 137-144, 2002; Ranson, M. and M. X Sliwkowski, Oncology, 63 Suppl 1: 17-24 (2002), Johnson, P. and M. Glennie, Semin. Oncol., 30: 3-8 (2003), Plosker, G. L. and D. P. Figgitt, Drugs, 63: 803-843 (2003) and Ross, J. S., et al., Am. J. Clin. Pathol., 119: 472-485 (2003)). Based on their record of success, several other humanized mAbs are being developed and some, such as Erbitux™ (cetuximab) are apparently close to achieving final FDA approval. Nevertheless, antibody therapy is not uniformly effective, even in patients whose tumors express a high surface density of the target tumor antigen. Effector mechanisms thought to cause tumor regression are variable and particularly include inhibition of growth factor activity, as well as antibody-dependent cell-mediated cytotoxicity (ADCC). Complement-dependent cytotoxicity (CDC) has less frequently been identified as an effector mechanism and it remains somewhat controversial whether CDC contributes significantly to tumor regression. In vitro studies have shown that CDC is limited by membrane regulators of the complement system, such as CD55 and CD59, that are occasionally overexpressed on tumors. Moreover, the major complement-mediated effector mechanism used against microbial pathogens, C3-receptor-dependent phagocytosis and cytotoxic degranulation, is completely inactive against cancer. With the antitumor human IgG1-based mAbs that activate complement such as trastuzumab, rituximab, or cetuximab, a coating of iC3b is deposited on tumor cells that can be recognized by the leukocyte iC3b-receptor CR3 (Mac-1; CD11b/CD18; αMβ2-integrin). However, the triggering of CR3-dependent leukocyte (neutrophil, monocyte, macrophage, NK cell) mediated cytotoxicity requires that CR3 bind to both iC3b and binding to the lectin site. Since tumor cells do not express CR3-activating polysaccharides, they escape this protective mechanism effective against microbial pathogens.
An increasing awareness exists determining that effective destruction of tumors by the immune system requires a combination of effector mechanisms. Thus, a single vaccine, cytokine, or biological response modifier is unlikely to be successful in a majority of patients. For example, vaccines may elicit immune cytotoxic T lymphocytes and/or humoral antibody responses, but each has shortcomings. Antibodies are frequently ineffective because normal host cell proteins such as DAF, MCP, and CD59 inhibit complement-mediated cytotoxicity. Further, iC3b-opsonization of tumors does not, solely, recruit phagocytes or NK cells. Antibody-dependent cell-mediated immunity is thought to fail because the IgG density achieved on tumors is too low and antibody Fc fragment-mediated cytotoxicity is suppressed by NK cell recognition of tumor cell MHC class I. Cell-mediated immunity utilizing cytotoxic T lymphocytes has disadvantages as well, since tumors, as part of the metastatic process, often lose the major histocompatability complex molecules required for antigen presentation. Therefore, a need exists for antitumor therapy that avoids the shortcomings discussed above.